B-cell lymphoma-2 (BCL-2) is an anti-apoptotic protein frequently upregulated in cancer enabling cell survival despite oncogenic stress. The resulting BCL-2 dependence sensitizes it to venetoclax, an FDA-approved drug used to treat chronic lymphocytic leukemia. However, treatment-induced mutations in BCL-2 and other resistance mechanisms frequently limit venetoclax efficacy. While it has been established that mutations G101V and D103Y, located in the binding site, disrupt BCL-2 interaction with venetoclax, we still fail to understand how mutations, especially those outside this region, result in resistance. Here, we performed a comprehensive comparison of the biochemical effects of frequently occurring cancer-associated BCL-2 mutations inside and outside the venetoclax-binding site. We found that G101V and D103Y exhibited a double effect by disrupting venetoclax interaction while increasing the binding and inhibition of specific proapoptotic proteins. In contrast, mutation V156D blocked venetoclax-binding, likely through allosteric effects reaching the binding site, without altering the inhibition of proapoptotic proteins. Other mutations, like A113G, R129L, and R139H, did not alter venetoclax-binding in solution, but could not efficiently be displaced from their interaction with proapoptotic proteins by venetoclax in their native mitochondrial environment in cells. Together, our work reveals and characterizes A113G, R129L, R139H, and V156D as novel BCL-2 VEN resistance mutations thereby showing that cancer-related BCL-2 mutations can alter venetoclax killing efficacy through different mechanisms that impact/correlate with their aggressiveness/prognosis in cancer. It also further validates the already known VEN resistance mutations G101V and D103Y.
Aufdermauer et al. (Sun,) studied this question.